The effect of the VOs (MIC) on E coli in NB over time

One o f the factors affecting the effectiveness o f antimicrobial agents is the length of contact time o f the compound in the microbial environment and hence with the microbial membrane. The effects o f the VOs by time were measured following a standardised procedure and the colonies were counted at each time point. The overnight broth colonies (3.5 X 10®) were calculated and the colonies in 25mL were 1.4 x 10^. The results were converted to Log. 10 bases (Table 5.3), showing the decrease o f colonies by time (Fig.

5.1).

Effect of thyme and thymbra VOs on E coli in nutrient broth

8 7 6

o 4 3 2 1 0

0 20 4 0 6 0 80 100 120 140

Time (min)

— thyme —m— thymbra

Fig. 5.1. Inhibitory effect o f thyme and thymbra VOs (MIC) on E. coli in nutrient broth over a time period of 120 minutes.

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Table 5.3. Inhibitory effect o f thyme and thymbra VOs on E. cloi growth in nutrient broth by time

Thyme VO 0.015 % Thymbra VO 0.02 %

Time Log. 10 cfu/mL Log. 10 cfu/mL

0 7.1 7.1

5 5.1 5.55

10 4.04 5.04

20 3.9 4.53

30 3.85 4.4

60 3.1 4.25

120 2.1 3.9

Thyme and thymbra VOs reduced the E. coli colonies count by 5 log and > 3 log, respectively, in two hours. The inhibitory effect o f the herbal VOs was mainly due to their major phenolic monoterpene thymol and carvacrol and this has been confirmed by researchers (Kunle et al. 2003, Helander et al. 1998, Friedman et al. 2002 and Olasupo et al. 2003). Carvacrol is known to disrupt the outer cell membrane o f E. coli (Helander et ah, 1998) and it was suggested that it acts as a proton exchanger by which reducing the pH across the cytoplasmic membrane resulting in the collapse o f the proton motive force and depletion o f the ATP pool resulting finally in cell death (Ultee et a l, 1999, 2002). In a study carried out by Di Pasqua et al. (2007) in which a number o f pathogens including E. coli were treated with a variety o f phenolic compounds (including thymol and carvacrol), the antimicrobial action o f the compounds occurred in the membrane leading to an alteration o f the fatty acid profiles as well as the modification or disintegration o f the cell structure. The mechanism o f action o f carvacrol and thymol at 200 mg 1'^ against E. coli has been attributed to their ability to permeabilize and depolarize the cytoplasmic

membrane (Xu, et al. 2008). Thymol and its isomer carvacrol, appear to make the cell membrane permeable (Lamber et ah, 2001), both are able to disintegrate the outer membrane o f Gram-negative bacteria by releasing lipopolysaccharides and thus increasing the permeability o f the cytoplasmic membrane to ATP, leading to leakage o f ions and other cell contents (Lambert et al. 2001; Utlee et a l, 2002).

5.3.4 The effect of VOs on E coli in milk over time 5.3.4.1 The effect o f VOs on E coli in whole milk

Milk is an ubiquitous food system, presented for public consumption in different forms in terms o f both compositional content and prior heat treatment. Milk is a multi component system, and in addition to water it contains protein, fat and carbohydrate (lactose). In this study, the effect o f the VOs on the inhibition o f E. coli were tested in whole milk, skimmed milk (to investigate the effect o f reducing the fat content) and decaseinated skimmed milk thus reducing the fat and protein present, compared to whole milk.

When the VOs were added at their MIC and MCC obtained for nutrient to whole milk inoculated with E. coli, none o f the VOs showed inhibitory activity. When the concentrations were increased from 0.015 and 0.02 % increased up to 0.5 % there was also no inhibitory activity o f the VOs. These results suggest that the VOs are not suitable preservatives for whole milk, and this may be due to the presence o f fat and protein in the whole milk. The VOs are composed mainly o f terpenoids which are highly lipophilic and will dissolve in the fat present in the milk; Furthermore, the phenolic compounds are likely to react with the milk proteins, particularly casein (Bartolomé et al. 2008), resulting in deactivation o f the phenolic compounds E. coli (Table 5.4).

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Table 5.4. Effects o f various concentrations o f thyme and thymbra VOs on whole milk inoculated with E coli.

0.05% 0.10% 0.25% 0.50%

Time thyme thymbra Thyme Thymbra Thyme Thymbra Thyme Thymbra

0 7.245 7.25 7.2 7.2 7.2 7.2 6.85 6.85

5 7.25 7.25 7.27 7.414 6.89 7.079 7.72 6.86

10 7.3 7.28 7.26 7.398 7.02 7.2 6.85 6.79

20 7.3 7.3 7.27 7.735 7.19 7.12 6.85 6.9

30 7.3 7.27 7.14 7.367 6.92 7.22 6.92 7.01

60 7.28 7.3 7.27 7.34 7.16 7.037 6.9 6.9

120 7.36 7.38 7.27 7.4 7.05 6.995 6.05 7.04

5.S.4.2. Effects o f thyme and thymbra VOs on E. coli in skimmed milk

To reduce the impact o f fat in the milk, thyme and thymbra VOs were tested in skimmed milk at the MIC and MCC concentrations that were obtained from the broth experiments.

Here, the effects o f the VOs on E. coli were attenuated possibly due to the presence o f proteins present in the milk (casein and whey). The concentrations o f the VOs were therefore increased to 0.03 % and 0.04 %, for thyme and thymbra, respectively. In the meantime the VOs (0.3 and 0.4 %) were tested on diluted E coli colony culture (10^

cfu.mL'^) to evaluate the effects o f colony concentration on VO activity, however, no signs o f activity were observed. Interestingly when higher VO concentrations were used the bacteriostatic and bactericidal were 0.05 % and 0.1 % determined, for thyme and thymbra, respectively (Table 5.5).

Table 5.5. Effect o f thyme and thymbra VOs at (0.03 + 0.04) %, 0.05 % and 0.1 % over time on E coli growth inoculated to skimmed milk.

0.03% 0.04% 0.05% 0.05% 0.10% 0.1%

Thyme and thymbra VOs (0.05 %) showed almost comparable bacteriostatic activity for E. coli in skimmed milk, reducing the bacterial load by 1.5 log. and the VOs (0.1 %) completely inhibited E. coli growth after 30 minutes (Fig. 5.2).

Effects of thyme and thymbra VOs (0.03 + 0.04, 0.5, 0.1) % on Ecoli in skimmed milk

— Thyme0.03 ■thymbra-0.04 thyme-0.05 —X— thymbra-0.05 —* — thyme-0.1 —#— thymbra-0.1

Fig. 5.2. Graphical illustration of the effect o f concentrations (0.03 + 0.04) %, 0.05 % and 0.01 % o f thyme and thymbra VOs on E coli in skimmed milk, showing static (0.05 %) and cidal (0.1 %) effects.

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While the VOs o f thyme and thymbra showed bactericidal activity against E. coli in broth at 0.02 % and 0.025 % (Table 5.2), the VOs were required at a higher concentration in skimmed milk to achieve the same effect. This is in line with the observations o f Shelef (1983) and Smid and Gorris (1999) who reported that in general a greater concentration than MIC o f VOs were needed in foods. Researchers used VOs in different food systems and the ratio required was approximately two fold in semi-skimmed milk (Karatzas et al, 2001) and 50 fold in soup (Ultee and Smid 2001).

In this study, the increase in VOs concentration can be attributed to the role o f milk proteins which binds to excess VOs MIC added to the skimmed milk. Fares et a l (1998) reported protein-phenol complex are formed through the binding sites o f the aromatic nuclei and the hydroxyl group o f the phenolic compounds, as both hydrogen bonding and hydrophobic interaction are the principal attractive forces between protein and phenolic groups (Wu et a l, 2008). The protein binding probably played a role when VOs were tested on E. coli at 10"^ dilution (2.2.3), the VOs (0.03 % and 0.04 %) showed no effect on E coli growth inhibition. To eliminate the possible role o f casein in the skimmed milk, casein was precipitated.

5.3.4.3 Casein precipitation

To eliminate the role o f casein in the skimmed milk, casein was precipitated. Following casein removal, the remainder o f the solution was considered to contain only whey proteins, bovine serum albumin and lactose. When tested the VOs, thyme VO (0.03 %) and thymbra VO (0.04 %) showed bacterial reduction by over 3 logs after 5 minutes, and

they completely inhibited E. coli growth after 30 and 60 minutes, respectively (Table 5.6.). These results indicate possible neutrality of lactose and also that the VOs are not bound by the whey proteins present in the milk solution, so that the free VOs molecules could act against the E. coli in the solution.

Effects of thyme and thymbra VOs (0.03 and 0.04) % on Ecoli in decaseinated skimmed rriik

1 0 . 0 0 n

To confirm the binding properties o f casein and BSA with thyme VOs, a broth was prepared that contained (3.4 % protein) sodium caseinate and bovine serum albumin in ratio o f 80 : 20. In this assessment, thyme VO (0.03 %) showed no inhibitory activity against E. coli (Table 5.7) and the outcome was comparable to that o f the original skimmed milk (2.2.5).

Table 5.7. Effect o f thyme VO (0.03 %) on E coli in reconstructed broth with albumin and casein

Time

Log.cfu/ml Albumin

Log.cfu/ml Casein

0 72 8 7.28

5 7.34 &55

10 7.24 7.26

20 7.34 7.02

30 7.42 7.02

60 7.46 6.89

120 7.46 6.64

These results suggest the binding capacity o f sodium caseinate (80 %) and BSA (20 %), a major and minor proteins in milk to the VOs thereby inhibiting their action against the E.

coli inoculated to the broth. Other phenolic compounds such as vanillin have also been shown to have strong interaction with BSA through hydrophobic interaction and sodium caseinate through hydrogen bonding (Chobpattana, et al. 2002).

The antimicrobial effects o f essential oils have been assessed in an array o f foods including meat, fish, dairy, vegetables, rice and fruit among which thyme oil and carvacrol have been extensively used. For instance carvacrol at 2-3 mM has been used in semi-skimmed milk; another preservative comprising 50 % EOs (rosemary, sage and citrus) has been used in soft cheese; clove, cinnamon, cardamom and peppermint oil (0.005 - 0.5 %) have been used in milk before fermentation for yoghurt production; and clove oil (0.5 -1 %) has been used in Mozarella cheese (Burt, 2004).

The significance o f this study lies in the possible application o f the thyme and thymbra VOs in a range o f foods. Clearly, however, the addition o f these oils to foods needs to be carefully considered in terms o f aroma and organoleptic compatibility with the food items. The demand for a healthier low-fat diet, combined with the trend for natural and green products (Smid and Gorris, 1999) and a lower usage o f synthetic compounds o f less impact on environment and the world wide call for reduction in salt consumption (WHO, 2002), require careful development o f safe product . The use of volatile oils in skimmed milk and its products could be considered for increasing microbial quality.

Further, the application o f thyme and thymbra VOs as food preservatives in low fat and protein content, products such as soft drinks including fruit juices needs more research in terms o f effectiveness and sensory acceptability.

5.4 Conclusion

This study demonstrated the antimicrobial properties o f Thymus syriaciis and Thymbra spicata volatile oils against a broad range o f microorganisms. It was observed that the

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MIC (0.015 % and 0.02 %) and MCC (0.02 % and 0.025 %) o f thyme and thymbra VOs respectively against E coli in nutrient broth. Further, both VOs showed bacteristatic at (0.05 %) and bactericidal (0.1 %) effects against E. coli in skimmed milk. Milk protein was found to have an adverse impact on the antibacterial effects o f the VOs used in whole milk and skimmed milk, whereas lactose was neutral when VOs were added to decaseinated skimmed milk. Both herbal VOs may be considered as food preservatives and could be applied to a range o f foods after an analysis o f their organoleptic effects in foods.